No attempt was made in our studies to differentiate the actions of any single component in CITRICIDAL®  from the intact product. All studies were approached from the view point of CITRICIDAL®  representing a product and all work was done on the product.

The test organisms used in these studies included: Salmonella sps., E. coli, Listeria monocytogenes, Candida albicans, Aspergillus parasiticus and Penicillium cyclopium.

In regard to CITRICIDAL liquid, significant progress has been made in our evaluation of this product as a disinfectant. Numerous studies have been conducted and a brief statement of the methodology with results follows. As indicated by the data, there is great potential for the development of this product as a disinfectant. This is based on the following: (1) the toxicological data indicates that this product and the active ingredient posses very low toxicity. This is important because most disinfectants that are currently used in either animal or human environments have moderate to high toxicity and extreme care must be exercised when these products are used. The lack of any significant toxicological properties of CITRICIDAL is also impressive when one views the efficacy data where extremely small concentrations of the product can be used with marked beneficial results. (2) In view of the reports discussed, the wide spectrum of activity that CITRICIDAL offers (antiviral, antibacterial, both gram- and gram+, antimycotic and antiprotozoan) will undoubtedly aid in its acceptability. (3) The fact that this product has a very pleasant aroma will aid in the overall acceptability. When used in the laboratory, comments pertaining to the "fresh" smell have been numerous. This may be considered a subtle point, however, we feel that it is important.

MATERIALS AND METHODS:

CITRICIDAL liquid was obtained from Bio/Chem Research Co. This solution was of rather high viscosity with a slight yellow color. The product was transparent with no detectable sediment. Preparation of various dilutions of the stock solution (100%) indicated that the solution was readily and completely water soluble. The aroma was pleasant.

In experiment #1, various dilutions of the concentrate were prepared. The concentrations of CITRICIDAL were 0, 0.125, 0.25, 0.50 and 2.0 ounces/gallon of dilutent. (NOTE: the 0.5 oz/gallon dilution is equivalent to approximately 3.9 ml/1OOOml). The dilatants used in these studies was either distilled water or distilled water containing 2.5% egg white. The egg white was used as a source of primarily protein to aid in the evaluation of CITRICIDAL in the presence of a high concentration of "organic matter" in comparison to CITRICIDAL in the absence of any proteinaceous material or minerals.

Three (3) ml of each concentration and dilutent combination of CITRICIDAL was added to sterile test tubes equipped with screw caps. Next, a suspension of a bacterial culture containing approximately 10 bacterial cells was added to each test tube. The organisms used were E, coli, Salmonella sps. or Staphyloccocus aureus. All of the bacterial species were obtained from field cases of disease of poultry. The E. coli was isolated from a systemic infection, the Salmonella from the intestinal tract and the S. aureus from an infected joint. All organisms are most likely pathogenic with at least moderate virulence. No serotyping was conducted on any of the organisms.

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Of perhaps more importance is the impression that is apparent from the data shown in Table #2. The presence of an extremely high content of protein (2.5% egg white) did not change the activity of CITRICIDAL disinfectant toward the three bacterial species used. There was no detectable change in the activity of this disinfectant toward the E. coli or Salmonella. A slight increase (from 5 to 10 minutes) to kill all the Staphylococus was measured, however due to this small magnitude of increase, this is of questionable significance.

Although not included in this report, preliminary studies with CITRICIDAL also indicate that this disinfectant poses good antifungal activity. This should also be kept in mind when considering the attributes of this product.

CITRICIDAL has great potential as a disinfectant with the previously determined low toxicity coupled with the excellent antibacterial activity suggests numerous applications of this product. The data in this report must be repeated before recommendations can be made as to the appropriate dilution of CITRICIDAL that should be used in practical application, however indications are that the "use rate" will be low. Additional, studies are planned to better define these levels (i.e. minimal inhibitory concentrations in a variety of media). The apparent refactory nature of this disinfectant to protein is intriguing and additional studies are currently underway to gain a better understanding of this aspect of CITRICIDAL.

Note:  No viable organisms were detected after 5 minutes at concentrations of Citricidal in excess of 0.125 oz/gallon

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Note:  No viable organisms present in Citricidal concentrations above 0.1235 oz. gallon after 10 minutes

NG:  No Growth